scholarly journals “Self-inactivating” rabies viruses are susceptible to loss of their intended attenuating modification

2019 ◽  
Author(s):  
Makoto Matsuyama ◽  
Lei Jin ◽  
Thomas K. Lavin ◽  
Heather A. Sullivan ◽  
YuanYuan Hou ◽  
...  
Keyword(s):  
Viral Vectors ◽  
Deletion Mutant ◽  
First Generation ◽  
Great Majority ◽  
Viral Particles ◽  
New Form ◽  
Transsynaptic Tracing ◽  
Circuit Function ◽  
Neuronal Physiology

SUMMARYAn article in Cell reported a new form of modified rabies virus that was apparently capable of labeling neurons “without adverse effects on neuronal physiology and circuit function” but that nevertheless was able to spread between neurons as efficiently as the widely-used first-generation deletion-mutant (ΔG) rabies viral vectors. The new “self-inactivating” rabies (“SiR”) viruses differed from first-generation vectors only by the addition of a destabilization domain to the viral nucleoprotein. We noticed that the transsynaptic tracing results from that article appeared inconsistent with the strategy described in it: specifically, the viruses were able to spread between neurons even in the absence of the exogenous protease that was meant to be required. We hypothesized that the viruses used were actually mutants that had lost the intended addition to the nucleoprotein, making them de facto first-generation viruses. We obtained samples of two SiR viruses from the authors and show here that the great majority of viral particles in both the “SiR-CRE” and “SiR-FLPo” samples were mutants that had lost the intended modification, consistent with our hypothesis. We also found that SiR-CRE killed 70% of infected neurons in vivo within two weeks, consistent with the prediction that mutants without the intended modification would share the toxic phenotype typical of first-generation rabies viral vectors. We hypothesize that the same or similar mutations were present in the viruses used in the original article and that this explains the paradoxical reported findings. While it may be possible to successfully make SiR viral preparations that are not dominated by such mutants, and while it may also be possible that such intact SiR viruses are indeed nontoxic to neurons, we predict that it will not be possible to replicate the transsynaptic tracing results from the original paper unless using mutants similar to the ones that we report here.

scholarly journals Monosynaptic tracing success depends critically on helper virus concentrations

2019 ◽  
Author(s):  
Thomas K. Lavin ◽  
Lei Jin ◽  
Nicholas E. Lea ◽  
Ian R. Wickersham
Keyword(s):  
Viral Vectors ◽  
Deletion Mutant ◽  
First Generation ◽  
Viral Vector ◽  
Helper Virus ◽  
Neuronal Population ◽  
Wild Type ◽  
Practical Guidelines ◽  
Mouse Lines ◽  
Wild Type Control

ABSTRACTMonosynaptically-restricted transsynaptic tracing using deletion-mutant rabies virus has become a widely used technique in neuroscience, allowing identification, imaging, and manipulation of neurons directly presynaptic to a starting neuronal population. Its most common implementation is to use Cre mouse lines in combination with Cre-dependent “helper” adeno-associated viral vectors (AAVs) to supply the required genes to the targeted population before subsequent injection of a first-generation (ΔG) rabies viral vector. Here we show that the efficiency of transsynaptic spread and the degree of nonspecific labeling in wild-type control animals depend strongly on the concentrations of these helper AAVs. Our results suggest practical guidelines for achieving good results.

Cell Biological and Biophysical Aspects of Lipid-mediated Gene Delivery

2006 ◽  
Vol 26 (4) ◽  
pp. 301-324 ◽  
Author(s):  
N. Madhusudhana Rao ◽  
Vijaya Gopal
Keyword(s):  
Gene Delivery ◽  
Viral Vectors ◽  
First Generation ◽  
Cationic Lipid ◽  
Cationic Lipids ◽  
Biological Knowledge ◽  
Effective Strategies ◽  
Hydrophilic Region

Cationic lipids are conceptually and methodologically simple tools to deliver nucleic acids into the cells. Strategies based on cationic lipids are viable alternatives to viral vectors and are becoming increasingly popular owing to their minimal toxicity. The first-generation cationic lipids were built around the quaternary nitrogen primarily for binding and condensing DNA. A large number of lipids with variations in the hydrophobic and hydrophilic region were generated with excellent transfection efficiencies in vitro. These cationic lipids had reduced efficiencies when tested for gene delivery in vivo. Efforts in the last decade delineated the cell biological basis of the cationic lipid gene delivery to a significant detail. The application of techniques such as small angle X-ray spectroscopy (SAXS) and fluorescence microscopy, helped in linking the physical properties of lipid:DNA complex (lipoplex) with its intracellular fate. This biological knowledge has been incorporated in the design of the second-generation cationic lipids. Lipid-peptide conjugates (peptoids) are effective strategies to overcome the various cellular barriers along with the lipoplex formulations methodologies. In this context, cationic lipid-mediated gene delivery is considerably benefited by the methodologies of liposome-mediated drug delivery. Lipid mediated gene delivery has an intrinsic advantage of being a biomimetic platform on which considerable variations could be built to develop efficient in vivo gene delivery protocols.

scholarly journals Acquisition of Complement Resistance through Incorporation of CD55/Decay-Accelerating Factor into Viral Particles Bearing Baculovirus GP64

2010 ◽  
Vol 84 (7) ◽  
pp. 3210-3219 ◽  
Author(s):  
Yuuki Kaname ◽  
Hideki Tani ◽  
Chikako Kataoka ◽  
Mai Shiokawa ◽  
Shuhei Taguwa ◽  
...  
Keyword(s):  
Envelope Protein ◽  
Viral Vectors ◽  
Molecular Mechanisms ◽  
Insect Cells ◽  
Decay Accelerating Factor ◽  
Complement Regulatory Proteins ◽  
Complement Resistance ◽  
Viral Particles ◽  
Lentivirus Vectors

ABSTRACT A major obstacle to gene transduction by viral vectors is inactivation by human complement in vivo. One way to overcome this is to incorporate complement regulatory proteins, such as CD55/decay accelerating factor (DAF), into viral particles. Lentivirus vectors pseudotyped with the baculovirus envelope protein GP64 have been shown to acquire more potent resistance to serum inactivation and longer transgene expression than those pseudotyped with the vesicular stomatitis virus (VSV) envelope protein G. However, the molecular mechanisms underlying resistance to serum inactivation in pseudotype particles bearing the GP64 have not been precisely elucidated. In this study, we generated pseudotype and recombinant VSVs bearing the GP64. Recombinant VSVs generated in human cell lines exhibited the incorporation of human DAF in viral particles and were resistant to serum inactivation, whereas those generated in insect cells exhibited no incorporation of human DAF and were sensitive to complement inactivation. The GP64 and human DAF were detected on the detergent-resistant membrane and were coprecipitated by immunoprecipitation analysis. A pseudotype VSV bearing GP64 produced in human DAF knockdown cells reduced resistance to serum inactivation. In contrast, recombinant baculoviruses generated in insect cells expressing human DAF or carrying the human DAF gene exhibited resistance to complement inactivation. These results suggest that the incorporation of human DAF into viral particles by interacting with baculovirus GP64 is involved in the acquisition of resistance to serum inactivation.

scholarly journals Long-term labeling and imaging of synaptically-connected neuronal networks in vivo using nontoxic, double-deletion-mutant rabies viruses

2021 ◽  
Author(s):  
Lei Jin ◽  
Heather A. Sullivan ◽  
Mulangma Zhu ◽  
Thomas K. Lavin ◽  
Makoto Matsuyama ◽  
...  
Keyword(s):  
Viral Vectors ◽  
Deletion Mutant ◽  
Neuronal Networks ◽  
Second Generation ◽  
Two Photon ◽  
Double Deletion ◽  
Viral Genes ◽  
Photon Imaging

SummaryThe highly specific and complex connectivity between neurons is the hallmark of nervous systems, but techniques for identifying, imaging, and manipulating synaptically-connected networks of neurons are limited. Monosynaptic tracing, or the gated replication and spread of a deletion-mutant rabies virus to label neurons directly connected to a targeted population of starting neurons1, is the most widely-used technique for mapping neural circuitry, but the rapid cytotoxicity of first-generation rabies viral vectors has restricted its use almost entirely to anatomical applications. We recently introduced double-deletion-mutant second-generation rabies viral vectors, showing that they have little or no detectable toxicity and are efficient means of retrogradely targeting neurons projecting to an injection site2, but they have not previously been shown to be capable of gated replication in vivo, the basis of monosynaptic tracing. Here we present a complete second-generation system for labeling direct inputs to genetically-targeted neuronal populations with minimal toxicity, using double-deletion-mutant rabies viruses. Spread of the viruses requires complementation of both of the deleted viral genes in trans in the starting postsynaptic cells; suppressing the expression of these viral genes following an initial period of viral replication, using the Tet-Off system, reduces toxicity to the starting cells without decreasing the efficiency of viral spread. Using longitudinal two- photon imaging of live monosynaptic tracing in visual cortex, we found that 94.4% of all labeled cells, and an estimated 92.3% of starting cells, survived for the full twelve-week course of imaging. Two-photon imaging of calcium responses in labeled networks of neurons in vivo over ten weeks showed that labeled neurons’ visual response properties remained stable for as long as we followed them. This nontoxic labeling of inputs to genetically-targeted neurons in vivo is a long-held goal in neuroscience, with transformative applications including nonperturbative transcriptomic and epigenomic profiling, long-term functional imaging and behavioral studies, and optogenetic and chemogenetic manipulation of synaptically-connected neuronal networks over the lifetimes of experimental animals.

Electrostatic Coating of Viral Particles for Gene Delivery Applications in Muscular Dystrophies: Influence of Size on Stability and Antibody Protection

2021 ◽  
pp. 1-11
Author(s):  
Marta Guerra-Rebollo ◽  
María Stampa ◽  
Miguel Ángel Lázaro ◽  
Anna Cascante ◽  
Cristina Fornaguera ◽  
...  
Keyword(s):  
Electrostatic Interaction ◽  
Viral Vectors ◽  
Dystrophin Gene ◽  
Muscular Dystrophies ◽  
Antibody Neutralization ◽  
Adeno Associated Virus ◽  
In Vivo Biodistribution ◽  
Biodistribution Studies ◽  
Viral Particles

Background: Duchenne Muscular Dystrophy (DMD) is one of the most common muscular dystrophies, caused by mutated forms of the dystrophin gene. Currently, the only treatment available is symptoms management. Novel approximations are trying to treat these patients with gene therapy, namely, using viral vectors. However, these vectors can be recognized by the immune system decreasing their therapeutic activity and making impossible a multidose treatment due to the induction of the humoral immunity following the first dose. Objective: Our objective is to demonstrate the feasibility of using a hybrid vector to avoid immune clearance, based on the electrostatic coating of adeno-associated virus (AAVs) vectors with our proprietary polymers. Methods: We coated model adeno-associated virus vectors by electrostatic interaction of our cationic poly (beta aminoester) polymers with the viral anionic capsid and characterized biophysical properties. Once the nanoformulations were designed, we studied their in vivo biodistribution by bioluminescence analysis and we finally studied the capacity of the polymers as potential coatings to avoid antibody neutralization. Results: We tested two polymer combinations and we demonstrated the need for poly(ethylene glycol) addition to avoid vector aggregation after coating. In vivo biodistribution studies demonstrated that viral particles are located in the liver (short times) and also in muscles (long times), the target organ. However, we did not achieve complete antibody neutralization shielding using this electrostatic coating. Conclusions: The null hypothesis stands: although it is feasible to coat viral particles by electrostatic interaction with a proprietary polymer, this strategy is not appropriate for AAVs due to their small size, so other alternatives are required as a novel treatment for DMD patients.

THE ROLE OF POLYETHYLENIMINE IN ENHANCING PERFORMANCE OF GLUTAMATE BIOSENSORS

2018 ◽  
Vol 8 (3) ◽  
pp. 36-41
Author(s):  
Diep Do Thi Hong ◽  
Duong Le Phuoc ◽  
Hoai Nguyen Thi ◽  
Serra Pier Andrea ◽  
Rocchitta Gaia
Keyword(s):  
First Generation ◽  
Good Reproducibility ◽  
Complex Matrices ◽  
Long Term Stability ◽  
In Vitro Characterization ◽  
Glutamate Oxidase

Background: The first biosensor was constructed more than fifty years ago. It was composed of the biorecognition element and transducer. The first-generation enzyme biosensors play important role in monitoring neurotransmitter and determine small quantities of substances in complex matrices of the samples Glutamate is important biochemicals involved in energetic metabolism and neurotransmission. Therefore, biosensors requires the development a new approach exhibiting high sensibility, good reproducibility and longterm stability. The first-generation enzyme biosensors play important role in monitoring neurotransmitter and determine small quantities of substances in complex matrices of the samples. The aims of this work: To find out which concentration of polyethylenimine (PEI) exhibiting the most high sensibility, good reproducibility and long-term stability. Methods: We designed and developed glutamate biosensor using different concentration of PEI ranging from 0% to 5% at Day 1 and Day 8. Results: After Glutamate biosensors in-vitro characterization, several PEI concentrations, ranging from 0.5% to 1% seem to be the best in terms of VMAX, the KM; while PEI content ranging from 0.5% to 1% resulted stable, PEI 1% displayed an excellent stability. Conclusions: In the result, PEI 1% perfomed high sensibility, good stability and blocking interference. Furthermore, we expect to develop and characterize an implantable biosensor capable of detecting glutamate, glucose in vivo. Key words: Glutamate biosensors, PEi (Polyethylenimine) enhances glutamate oxidase, glutamate oxidase biosensors

The Synergy between CRISPR and Chemical Engineering

2019 ◽  
Vol 19 (3) ◽  
pp. 147-171
Author(s):  
Cia-Hin Lau ◽  
Chung Tin
Keyword(s):  
Viral Vectors ◽  
Chemical Engineering ◽  
Target Genes ◽  
New Technologies ◽  
Rapid Development ◽  
Genetic Circuits ◽  
Viral Packaging ◽  
Conditional Control ◽  
Logic Operations

Gene therapy and transgenic research have advanced quickly in recent years due to the development of CRISPR technology. The rapid development of CRISPR technology has been largely benefited by chemical engineering. Firstly, chemical or synthetic substance enables spatiotemporal and conditional control of Cas9 or dCas9 activities. It prevents the leaky expression of CRISPR components, as well as minimizes toxicity and off-target effects. Multi-input logic operations and complex genetic circuits can also be implemented via multiplexed and orthogonal regulation of target genes. Secondly, rational chemical modifications to the sgRNA enhance gene editing efficiency and specificity by improving sgRNA stability and binding affinity to on-target genomic loci, and hence reducing off-target mismatches and systemic immunogenicity. Chemically-modified Cas9 mRNA is also more active and less immunogenic than the native mRNA. Thirdly, nonviral vehicles can circumvent the challenges associated with viral packaging and production through the delivery of Cas9-sgRNA ribonucleoprotein complex or large Cas9 expression plasmids. Multi-functional nanovectors enhance genome editing in vivo by overcoming multiple physiological barriers, enabling ligand-targeted cellular uptake, and blood-brain barrier crossing. Chemical engineering can also facilitate viral-based delivery by improving vector internalization, allowing tissue-specific transgene expression, and preventing inactivation of the viral vectors in vivo. This review aims to discuss how chemical engineering has helped improve existing CRISPR applications and enable new technologies for biomedical research. The usefulness, advantages, and molecular action for each chemical engineering approach are also highlighted.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

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